ABSTRACT This is a revised application for the K23 award for Dr. Marcelle Nascimento, an Assistant Professor at the University of Florida, College of Dentistry (UFCD). Dr. Nascimento has been conducting clinical-translational research projects focusing on the alkali-producing capacity of bacteria colonizing the oral cavity and its role in oral health and dental caries. Dr. Nascimento aims to become a leading and independent researcher in the field of Cariology and to pursue the creation of novel therapeutic targets based on oral alkali production that may fundamentally change the way in which we assess caries risk and prevent caries. She has assembled an outstanding mentoring group comprised by Drs. Robert Burne (UFCD), Robert Primosch (UFCD) and Floyd Dewhirst (Forsyth Institute) and the consultant Dr. Jorge Frias-Lopez (Forsyth Institute) to insure the success of her career development and proposed research study. This K23 award will greatly facilitate her career development by providing the structure and the guidance for acquiring: (1) expertise on designing and conducting patient-oriented studies, (2) training on bioinformatics and microbiome data analysis, (3) experience and mentoring in requesting and managing federal funds, and (4) the time release and the resources to generate data and publications to support an RO1 application by the end of the award period. Alkali production by oral bacteria has a major impact on dental plaque biochemistry and metabolism in a manner that reduces the cariogenicity of oral biofilms. Although acid production by plaque bacteria is known as the direct cause of caries, it is noteworthy that increases in the proportions of aciduric organisms appear to occur at the expense of species that are less aciduric and commonly associated with oral health - many of which have been recognized as alkali-producing bacteria. Loss of alkali-producing bacteria from the biofilm populations may reduce the capacity of plaque to neutralize acids and slow the return of plaque pH to more neutral values. Clinical studies from Dr. Nascimento's research group offer significant support that a lack of alkali production by oral bacteria may be a critical determinant of caries risk in adults. Specifically, the bacterial populations from plaque and saliva of caries-free subjects have a greater capacity to produce ammonia from arginine, which is one of the predominant alkali-yielding substrates in the oral cavity, when compared to bacterial populations from caries-active subjects. Differences in the microbial composition and regulation of the alkali-producing systems may be critical factors affecting the capacity of oral samples from different subjects to metabolize arginine. Of the known alkali-generating systems by oral bacteria, the arginine deiminase system (ADS) is perhaps the single most significant pH homeostasis mechanism in plaque, and appears to contribute in major ways to resistance to caries. Through the arginine decarboxylase enzyme (ADC), arginine can also be catabolized into agmantine and then into ammonia by the agmatine deiminase system (AgDS). While novel and important findings on oral arginolytic activity have recently arisen from her clinical study with adults and preliminary study with children, the arginine catabolic potential of oral bacteria from children with different caries-status remains unexplored, in spite of the importance of this pathway to plaque pH homeostasis and caries. Likewise, fundamental questions remain with regards to the oral bacteria: (a) constitutional arginolytic capacity, (b) sensitivity to regulation of the ADS and AgDS, (c) constitutional acid resistance, and (d) gene content, as it correlates to the role of ADS and AgDS in the ecology of oral biofilms, health and disease. Accordingly, the goals of the proposed experimentation are: (i) to continue to investigate the relationship between oral alkali production from arginine and agmatine and caries experience in children, and (ii) to gain insights into the fundamental microbiology, ecology and metagenomics of supragingival ammonia-producing bacterial communities and their relationship to oral health and dental caries. These studies will provide essential baseline information for the development of new strategies for caries risk assessment and caries control that are based on modification of oral biofilm pH and ecology through alkali production.